Agonist-specific voltage sensitivity at the dopamine D2S receptor – Molecular determinants and relevance to therapeutic ligands
Highlights
► We investigate agonist-specific voltage sensitivity at the dopamine D2S receptor. ► Dopamine binding to receptor is decreased by membrane depolarization. ► Dopamine potency in G protein activation assay is decreased by depolarization. ► Specific interactions between dopamine and the receptor determine voltage sensitivity. ► Different therapeutic agonists are affected differently by D2S voltage sensitivity.
Introduction
The dopamine D2 receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is the main target for therapeutic drugs against several common neurological, neuroendocrinological, and psychiatric disorders such as Parkinson’s disease, hyperprolactinemia and schizophrenia (Kvernmo et al., 2008, Prabhakar and Davis, 2008, Seeman, 2006). Dopamine D2 receptor agonists make up an essential part of the pharmacological inventory for treatment, especially for the first two of these disorders. The dopamine D2 receptor is expressed as two distinct splice variants; D2L and D2S (long and short), differing by a stretch of 29 residues in the third intracellular loop (Usiello et al., 2000). D2S functions as an inhibitory auto- and heteroreceptor at dopamine, glutamate, and GABA terminals in the CNS, whereas D2L is considered to mediate the majority of postsynaptic responses to dopamine (Usiello et al., 2000).
Whereas GPCRs have not traditionally been regarded as sensitive to membrane potential, increasing evidence to this effect has accumulated in recent years (see Mahaut-Smith et al., 2008, Parnas and Parnas, 2010). Decreases in agonist binding affinity at Gi/o-coupled M2 muscarinic receptor and glutamate mGluR3 upon membrane depolarization were shown both by electrophysiological assays in Xenopus oocytes, using G-protein coupled inward rectifier potassium channel (GIRK) opening as readout of receptor activity, as well as by radiolabeled agonist binding experiments in Xenopus oocytes (Ben-Chaim et al., 2003, Ohana et al., 2006). Conversely, for Gq-coupled M1 muscarinic receptor and mGluR1 depolarization caused an increase in agonist binding affinity (Ben-Chaim et al., 2006, Ohana et al., 2006). In other studies of lysophosphatidic acid receptors expressed in oocytes, as well as P2Y1 and other Gq-coupled receptors in rat megakaryocytes, increases in agonist potency were observed upon depolarization (Martinez-Pinna et al., 2010, Gurung et al., 2008).
We recently demonstrated voltage sensitivity of both splice variants of the dopamine D2 receptor using the GIRK activation assay (Sahlholm et al., 2008a, Sahlholm et al., 2008b). It was further shown that this sensitivity has distinct consequences for the potencies of different agonists; whereas potencies of several D2 receptor agonists were reduced upon depolarization from −80 to 0 mV, the EC50s of other agonists were not significantly affected (Sahlholm et al., 2008c). Such agonist-specific effects of GPCR voltage sensitivity were also reported recently for the muscarinic M2 receptor (Navarro-Polanco et al., 2011).
Furthermore, Ben-Chaim et al. (2006) recorded charge movement within the M2 receptor, which correlated with the voltage-dependent shift in binding affinity, and Kupchik et al. (2011) as well as Navarro-Polanco et al. (2011) further demonstrated that this charge movement is affected by the presence of receptor ligands in a concentration-dependent, ligand-specific manner. These findings suggest that parts of the receptor itself move upon changes of the membrane potential; presumably, one or several charged, voltage-sensing residues move in response to voltage changes, and these movements are relayed to the ligand binding site of the receptor.
The D2 receptor orthosteric ligand binding site is relatively well characterized and much is known about the molecular basis of ligand–receptor interactions (Javitch, 1998, Floresca and Schetz, 2004). In light of the previous findings outlined above, we wished to explore further the relationships between agonist–receptor interactions and voltage-induced effects on agonist potency. We restricted our analysis to D2S, since most earlier mutational studies have involved this splice variant. However, since the D2L and D2S binding sites are identical at the level of amino acid sequence, our findings are likely applicable also to D2L.
Initially, we wanted to examine whether the mechanism behind the depolarization-induced potency decrease of dopamine at the D2S receptor indeed involves decreased dopamine binding at depolarized potentials. We also aimed to confirm that this voltage sensitivity can be observed upstream of effector proteins, at the level of G protein activation.
Secondly, we hypothesized that agonist-specific voltage sensitivity is dependent on differences in the interactions between ligand and receptor. Thus, we sought to identify ligand–receptor interactions responsible for the differential impact of receptor voltage sensitivity on different agonists at the D2S receptor by site-directed mutagenesis of residues in the ligand binding site, and by further variation of agonist structure (see Fig. 1).
Finally, given the prominent role for dopamine receptor agonists in therapy and as pharmacological tools, we wanted to investigate whether a range of clinically used D2 agonists would show any differences in terms of how they are affected by D2S voltage sensitivity.
Section snippets
Molecular biology
Human GIRK1 (Kir3.1) and GIRK4 (Kir3.4) cDNA (provided by Dr. Terence Hebert, University of Montreal, Canada) were in pCDNA3 (Invitrogen). cDNA encoding the human dopamine D2S receptor was in pGEM4Z (a gift from Dr. Marc Caron, Duke University, North Carolina; used for most oocyte experiments with the wt receptor) or in pXOOM (for oocyte experiments with mutant receptors, radioligand binding experiments and for all experiments involving mammalian cells). The dual-purpose vector, pXOOM, contains
Voltage sensitivity of [3H]dopamine binding
We previously demonstrated that depolarization reduces the potency of dopamine in activating GIRK channels via the dopamine D2S receptor (Sahlholm et al., 2008b, Sahlholm et al., 2008c). To test whether diminished agonist binding underlies this decrease in potency, binding of [3H]dopamine to oocytes expressing D2S receptors was measured at two membrane potentials. An example of 40 nM of [3H]dopamine binding to oocytes expressing D2S receptors and GIRK channels at −82 mV (ND96) and at 0 mV (high
Discussion
In the present study, we first demonstrate that the reduced potency of dopamine observed in the GIRK channel activation assay at depolarized potentials is correlated with a reduction of D2S receptor binding of radiolabeled dopamine. This is in line with previous findings concerning voltage sensitivity of muscarinic M2 and glutamate mGluR3 receptors (Ben-Chaim et al., 2003, Ohana et al., 2006). We further demonstrate that D2S receptor voltage sensitivity is observed also in mammalian cells, at
Conclusion
In the present work, we showed that the depolarization-induced decrease in dopamine potency observed at the level of GIRK channel activation correlates with a depolarization-induced decrease in dopamine binding and G protein activation. We also identified some of the ligand–receptor interactions which determine the effect of receptor voltage sensitivity on agonist potency, and demonstrated that a number of clinically used dopamine agonists differ in terms of the voltage sensitivity of their
Role of the funding source
The funding sources, listed under “Acknowledgments”, had no role in study design, in the collection, analysis, and interpretation of data, in manuscript writing, or in the decision to submit the manuscript.
Acknowledgments
The present work was supported by grants from the Swedish Research Council, and the Parkinson Foundation in Sweden, to P.Å., Åhlén-stiftelsen, the Swedish Society for Medical Research, Gålöstiftelsen, and Stiftelsen Lars Hiertas Minne, to K.S., and by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005, from Ministerio de Ciencia e Innovación, and ICREA Academia-2010 from the Catalan Institution for Research and Advanced Studies, to F.C. The authors wish to thank Dr. Hanna Parnas, The
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